Capillary electrophoresis (CE) is an analytical technique that uses large electrical potentials applied across narrow bore fused silica capillaries to separate ions in solution. In the applied electrical field, positive and negative ions migrate in solution towards the anode and cathode, respectively. In addition, an electroosmotic flow can also present during the CE processes, depending on the surface charge of the inner capillary wall, the pH, and the electrolyte composition.
Although CE gives excellent separation efficiencies, the small (<100 μm) capillary inner diameters give very short path lengths for optical detection methods. This, along with the small injection volumes used, leads to a concentration sensitivity that is often lower than that achievable using liquid chromatography. One attractive alternative to optical detection is mass spectrometry (MS), which in addition to providing sensitive detection gives additional separation in gas phase and structural information on the analytes. However, interfacing the two methods presents a number of challenges. In order to be analyzed by MS, the ions in solution during CE must be converted to gaseous ions. Additionally, in order to operate in an online fashion the outlet vial of a typical CE instrument must be replaced by another means of electrical contact that does not significantly reduce the separation resolution.
The most popular method to achieve this coupling is electrospray ionization (ESI), which was first proposed as a source of ions for mass analysis. The various teachings of Fenn et al. helped to demonstrate the potential of ESI for mass spectrometry. Since then, ESI has become one of the most commonly used types of ionization techniques due to its versatility, ease of use, and effectiveness for large biomolecules.
ESI involves applying a high electrical potential to a liquid sample flowing through a capillary. Droplets from the liquid sample become charged and an electrophoretic type of charge separation occurs. In positive ion mode ESI, positive ions migrate downstream towards the meniscus of the liquid at the tip of the capillary. Negative ions are repelled back towards the capillary, resulting in charge enrichment. Subsequent fissions or evaporation of the charged droplets result in the formation of single solvated gas phase ions. These ions are then transmitted to the aperture of the mass spectrometer for separation based on their mass to charge ratio and detection.
The challenge in CE-ESI-MS is that both the CE and ESI processes require stable electrical contact of the solution with an electrode at the capillary outlet without interruption of the electroosmotic flow from the CE separation. Many different interfaces have been proposed, however most suffer from issues of excessive sample dilution, loss of resolution, spray instability and/or fragility and cost of the interface. The interfaces proposed for CE-MS can be divided into two categories: those using an additional liquid flow which mixes with the CE eluent, and those which do not.
The first category, known as sheath-flow interfaces, was the most popular type of interface in the early years of CE-MS appplications and is also the design found in current commercial CE-ESI-MS systems. The flowing sheath liquid that surrounds the capillary terminus serves two purposes. The first is to establish electrical contact with the capillary solution in order to drive the CE separation and the ESI process. The second purpose is to modify the composition of the CE electrolyte to make it more compatible with ESI and MS detection. In addition, in the early stages of CE-MS development most interfaces were adapted to fit into existing LC-MS setups, which required much higher flow rates than those delivered by CE. Therefore, the sheath liquid also served to increase the liquid flow to levels comparable to those found in liquid chromatography.
Sheath-flow interfaces also can be further divided into two categories: those where the sheath liquid flow is coaxial with the separation capillary and mixes with the separation buffer at the capillary terminus, and those where the sheath liquid is added by means of a junction before the CE terminus. It has been demonstrated that coaxial sheath flow interfaces give improved performance over those with a liquid junction.
Although sheath-flow interfaces do allow for more diverse conditions to be used during the CE separation, the addition of the sheath liquid dilutes the samples and leads to a significant loss in sensitivity. Because the small injection volumes used in CE give a concentration sensitivity that is low to begin with, this additional loss is in many cases an unacceptable sacrifice. More recently, sheath-flow interfaces have been developed that use even lower flow rates (some less than 200 nL/min) (Wahl, J. H., et al., Attomole Level Capillary Electrophoresis-Mass Spectrometric Protein Analysis Using 5-μm-i.d. Capillaries. Analytical Chemistry, 1992. 64: p. 3194-3196; Olivares, J. A., et al., On-Line Mass Spectrometric Detection for Capillary Zone Electrophoresis. Analytical Chemistry, 1987. 59: p. 1231). One of these, the pressurized liquid junction, is similar to the original liquid junction design, however the junction is slightly wider (up to 300 μm) and is located in a pressurized reservoir of make-up liquid. The addition of pressure helps to prevent defocusing of the CE effluent in the gap region that would lead to reduced resolution. To prevent back-flow due to the pressure differential across the separation capillary the inlet vial must also be pressurized. The conductive make-up liquid establishes electrical contact between the background electrolyte (BGE) and the shared electrode, and also supplies a consistent flow to the electrospray tip in cases when the flow rate from CE is insufficient. The additional flow introduced in these ‘pressurized junction’ interfaces does add a dilution factor, however it is much less than in the case of more traditional sheath-flow interfaces.
A sheath-flow nanospray interface has also been developed using a coaxial arrangement of silica capillaries. The terminal end of the narrow separation capillary is coated with gold to create an electrical contact outside of the separation path. It is then inserted into a larger-diameter silica capillary with the end pulled to a taper. The coaxial capillary assembly is mounted in a standard ionspray interface. Sheath liquid is passed through the larger capillary and flows over the end of the separation capillary, carrying CE effluent to the tapered tip. The dilution factor with this arrangement is less than ½ and the total flow rate of the combined solutions is approximately 500 nL/min.
Another strategy for low volume sheath-flow electrospray interface uses a beveled tip to reduce the required flow rates for stable spray operation without significantly reducing the inner diameter of the emitter tip. One application of the beveled tip uses a novel mixing arrangement that is neither coaxial nor a traditional liquid junction. The CE effluent and sheath liquid are delivered to the emitter tip in parallel capillaries and mixing occurs directly at the emitter orifice.
Despite the dilution that is inherent to sheath-flow interfaces, they offer a number of important advantages. Because the solution exiting the interface is primarily made up of sheath liquid, it is possible to use a wider variety of background electrolytes or additives in the CE process that might otherwise be incompatible with ESI-MS. It is also advantageous to use the sheath-liquid to create electrical contact at the CE capillary terminus, as this keeps the electrolysis process away from the analyte path. Finally, sheath-flow interfaces are generally robust and well suited to commercialization.
Despite recent advances, sheath-flow interfaces have yet to match the sensitivity achievable with sheathless interfaces. Sheathless interfaces are often categorized by the number of pieces through which the liquid flow passes. The first and most common type of sheathless interface involves only a single section of capillary which acts as both the separation channel and the electrospray emitter. In fact, the very first demonstration of mass spectrometry as an online detector for capillary electrophoresis was reported by Olivares and coworkers in 1987 using an interface fabricated by vapour deposition of silver onto a capillary terminus protruding slightly from a metal sheath electrode. The deposited metal created contact between the sheath electrode and the CE electrolyte.
Several other conductive coating materials have been tested in addition to silver, including gold copper, nickel and graphite. Unfortunately coated tips have short lifetimes due to the high electrical fields acting on the metal coating at the tip. Generally they can only be used for a few days before the deterioration of the coating renders operation unstable. Stability may be improved by pre-treating the capillary surface or mixing different materials into the coating.
Peters son and coworkers explored the possibility of using a thin film of static liquid between the capillary tip and a metal sheath pulled back slightly from the capillary tip to establish electrical contact. It has also been demonstrated that CE-ESI-MS can be performed with no electrode whatsoever at the capillary terminus. In this case electrical contact is established through the space between the capillary tip and the grounded orifice of the mass spectrometer. Although this appears to offer an extremely simple solution to interfacing, the position of the capillary tip with respect to the mass spectrometer is critical and it is not possible to control the separation and spray voltages independently.
An alternative to coating the capillary tip is to insert a wire electrode into the capillary channel in order to make electrical contact. Several different means to this end have been tested. When larger inner diameter capillaries are used a thin wire electrode may be inserted into the end of the capillary channel or into a small hole drilled near the capillary terminus. However, this creates turbulence and reduces the resolving power of the CE separation. Turbulence can be reduced by using a hole filled with conductive gold epoxy rather than wire, however, as with any situation where electrolysis occurs within the separation channel this may lead to bubble formation inside the separation channel.
Another strategy for creating electrical contact is to split the liquid flow from the capillary so that a portion of the flow contacts an outside electrode, known as a split-flow interface. Splitting is achieved through a drilled hole or a small crack in a single capillary which serves both as the separation chamber and electrospray tip. While this does well at preserving the separation, the difficulty in this strategy lies in creating reproducible holes or cracks which give the desired split ratio between the two flow paths. An alternative process is the use of hydrofluoric acid to etch away sections of the outside surface of the fused silica capillary to the point where the capillary walls become porous. Electrical contact can then be made through the porous location of capillary wall, either by immersing the etched portion of the capillary in a buffer reservoir, or by inserting it into a metal sheath filled with a thin film of liquid. Although interfaces of this type have been shown to be quite successful, the production is unappealingly hazardous and the capillaries are extremely fragile.
In two-capillary sheathless interfaces, the ends of the separation capillary and a capillary acting as the spray tip are closely butted together at a junction. No additional flow is introduced through the junction however electrical contact is established through a surrounding electrolyte into which the terminal electrode is placed. Junctions have been constructed using microdialysis tubing, a metal sleeve connected to the power source, or a to align the two capillaries and to introduce contact with an electrode. Although these techniques offer the advantage of moving the location of the electrolysis process to the outside of the CE circuit, they are difficult to align in a way that will not decrease the separation resolution. Similarly it is also possible to join the separation capillary with a metal tip that acts as both the sprayer and electrode, however alignment and bubble production remain problematic.
It has been well documented that many organic solvents, salts and other additives commonly used in CE can have a negative impact on the ionization efficiency of analytes of interest. This can be resolved in part by the use of a sheath-flow or liquid-junction interface, which alters the composition of the CE effluent with a more compatible sheath liquid. Similar concepts also exist in liquid chromatography. For example, a modifying solution has been added to LC effluent to counteract the ionization suppression due to trifluoroacetic acid in the mobile phase. Adjustment of this type to the chemical environment of the analytes can significantly increase the detection sensitivity by optimizing ionization conditions.